The present invention relates to a purifying method for obtaining a high purity product of daunomycin which is an anthracycline antibiotic.
It is known that daunomycin (called as well daunorubicin) is obtained from a culture medium for ray fungus, and it is an anthracycline antibiotic which has a wide range anticancer spectrum against experimental tumor (refer to U.S. Pat. No. 3,616,242). As a matter of fact, it is widely used as a cancer chemotherapeutic agent for a clinical purpose.
Analogous anthracycline: antibiotics including daunomycin (hereinafter abbreviated as xe2x80x9cDMxe2x80x9d) are further purified by treating products roughly purified from the culture medium described above with a cation exchange resin (for example, Amberlite(trademark) IRC 50) and eluting them with a sodium chloride-containing aqueous solution or methanol containing sodium chloride (Japanese Patent Publication No. 44347/1974 and Japanese Patent Application Laid-Open No. 15880/1975). Further, published as well are methods comprising the steps of adsorbing the antibiotic described above onto a polymeric ion exchange resin (for example, Amberlite(trademark) ER 180) or a CM Sepharose resin (for example, Sepharose(trademark) CHB) and then eluting it with a mixture of acidic water and a polar solvent (Japanese Patent Application Laid-Open No. 118797/1984 and Japanese Patent Publication No. 39476/1992 originating therein, divisionally filed Japanese Patent Application Laid-Open No. 252291/1995, and U.S. Pat. No. 4,861,870 corresponding to them). Further, it is described as well in the latter official gazettes that a roughly purified anthracycline antibiotic is adsorbed onto an adsorptive porous synthetic resin carrier (for example, Amberlite(trademark) XAD2) at a pre-purifying step before treating with the polymeric ion exchange resin described above, and the antibiotic described above is then eluted with a mixture of water/methanol (5:1) (v/v).
It seems that DM having a fixed high purity can be obtained according to the conventional adsorbing and eluting techniques described above. However, the more the high purity product is tried to obtain, the more the recovery percentage of intended DM is reduced. In contrast with this, the more the recovery percentage is tried to enhance, the more the purity of the product can not be elevated. In particular, when trying to recover DM from a culture medium, purified DM tends to be accompanied with impurities (for example, those having a longer retention time than that of DM in HPLC analysis which shall be described later) which are difficult to be removed at purifying steps including crystallization subsequent thereto.
Accordingly, an object of the present invention is to provide a purifying method in which high purity DM can be obtained from starting crude DM at a high yield in a simple manner.
The present inventors have found that DM having a high purity can be obtained at a high yield by using an adsorptive (or hydrophobic) synthetic resin carrier which is used at a preliminary purifying step prior to treatment using an ion exchange resin (for example, Amberlite(trademark) ERI 180) for obtaining a high purity product and by selecting specific elution (or dissolution) conditions. It shall be quite surprising that the problems described above can be solved only by using a hydrophobic synthetic resin carrier which is merely used at a preliminary purifying step where an ion exchange resin is inevitably used in order to obtain a high purity product as described in, for example, Japanese Patent Application Laid-Open No. 118797/1984 described above.
Hence, according to the present invention, provided is a purifying method of daunomycin characterized by eluting daunomycin from a hydrophobic porous synthetic resin carrier onto which daunomycin is adsorbed from crude daunomycin with an aqueous solution which contains a water-miscible organic solvent and which is buffered to pH 2.5 to 6 and recovering purified daunomycin from the eluent.